Project Summary/Abstract: A hypervirulent Klebsiella pneumoniae (hvKp) pathotype is undergoing global dissemination. In contrast to the usual healthcare-associated epidemiology of classical K. pneumoniae (cKp) infections, hvKp causes tissue invasive infections in otherwise healthy individuals from the community. Infection often involves multiple sites that require source control (e.g. abscesses, necrotizing fasciitis) or locations that require site-specific therapy (e.g. endophthalmitis, meningitis). Initial strains of hvKp were antimicrobial susceptible, however, recently hvKp strains have been acquiring genes that encode extended-spectrum -lactamases and carbapenemases. The reverse direction of transfer also can occur. Recently an extensively drug-resistant (XDR) cKp strain from sequence type ST11 that had acquired part of a virulence plasmid present in hvKp was implicated as the cause of a lethal outbreak in an intensive care unit. The prospect of a hypervirulent XDR pathogen is extremely concerning. The goal of this proposal is to increase our limited understanding of the factors and generate initial insights into the mechanisms responsible for hvKp?s hypervirulent phenotype. This, in turn, will enable logical strategies to prevent or treat infections due to this true superbug. To accomplish this, we will utilize previously generated in vivo virulence data developed with accurately defined cohorts of hvKp and cKp strains. These studies identified four strain classes: 1- prototypical hvKp strains that possessed known virulence factors (VFs) and were fully virulent in vivo; 2- hvKp strains that possessed known VFs but were less virulent than prototypical hvKp strains in vivo; 3- prototypical cKp strains that did not possess known hvKp VFs and were significantly less virulent in vivo compared to prototypical hvKp strains; and 4- cKp strains that did not possess known hvKp VFs but were more virulent in vivo compared to prototypical cKp strains. These strain classes will be used as ?tools? to identify candidate genes that encode VFs or anti-VFs via genomic sequencing, sequence analysis and delineation of gene sets, and subsequent appropriate comparisons (aim 1). Putative novel or unrecognized VFs and anti-VFs will be prioritized, isogenic mutant and complemented derivatives will be generated, and these constructs will be assessed in in vivo pneumonia and systemic infection models (aim 2). The deliverables of this proposal will be the identification and in vivo validation of new or unrecognized VFs and/or lack of anti-VFs that contribute to hvKp?s hypervirulent phenotype.